Audio recording of this lecture

Previous or Next lecture

Deinococci, Chlamydia and Planctomycetes



With this lecture, we round out the traditional 13 ‘main’ Phyla of Bacteria. Two of these Phyla, the Chlamydiae and Planctomycetes, are specifically related. The third, Deinococcus/Thermus is an independent lineage and represent another relatively primitive deeply-branching lineage of the Bacteria.

Phylum Deinococci (Deinococcus/Thermus)



Taxonomy

• Phylum Deinococcus/Thermus
  • Class Deinococci
    • Order Deinococcales
      • Family Deinococcaceae
        • Genus Deinococcus
      • Family Trueperaceae
        • Genus Truepera
    • Order Thermales
      • Family Thermaceae
        • Genus Thermus
        • Genus Marinothermus
        • Genus Meiothermus
        • Genus Oceanothermus
        • Genus Vulcanothermus

About this phylum

This phylum contains only two well-known genera: Deinococcus and Thermus. These organisms are quite different phenotypically and phylogenetically, and each represent a small collection of closely-related, very similar species.

Deinococcus & relatives

Diversity
Deinococcus, the only genus in the Family Deincoccales, consists of 18 closely-related species and a collection of other partially characterized isolates. A second genus, Deinobacter, was previously represented by a single specie, D. grandis, which has been reclassified as a member of the genus Deinococcus. The only exception is an additional single specie, Truepera radiovictrix, which is more distantly-related to Deinococcus and shares both the thermophilic phenotype of Thermus and the radiation-resistant phenotype of Deinococcus.

Metabolism
The Deinococci are aerobic heterotrophs, and most are mesophilic. The most striking feature of these organisms is their extreme resistance to ionizing (gamma) radiation, but they are also extremely resistant to UV radiation, dessication, oxidizing agents and mutagens. The common thread is that these all cause damage to DNA, and in the most extreme cases double-stranded breaks. Deinococcus has very active DNA repair systems, and by keeping between 4 and 10 copies of the genome in each cell, homologous recombination can even be used to to reassemble the DNA after wholesale fragmentation by high-energy gamma-irradiation.

Morphology
The Deinococci are non-motile cocci, except for the rod-shaped D. grandis. Although they have a Gram-negative -type envelop, the peptidoglycan layer is very thick, and the outer membrane is covered in an S-layer, resulting in them typically staining Gram-positive. As a result of this and the fact that they are commonly pigmented (pink or red to purple or even black), they can easily be mistaken for Micrococcus. Cell division is unusual in Deinococci - instead of the cells pinching-off into 2 daughter cells, cells divide by forming a ‘septal curtain', which closes inward like the shutter on a camera, without changing the shape of the original cell. The division plane alternates by ca. 90° in the X and Y (but not Z) axes, resulting in tetrads or larger arrangements of cells in some species.

Habitat
Most species were isolated from irradiated samples, including foods supposedly sterilized by irradiation, cleanrooms (which us UV lights for ‘sterilization’), and nuclear reactor cooling pools, but the natural environment of these organisms is not known. They have be isolated sporadically from soils, sediments, sewage, and many dust-covered surfaces. This suggests that their resistance to radiation might be a by-product of their evolved resistance to desiccation, which also induces double-stranded breaks in DNA. It has ben suggested that the natural habitat of this organism might be the water droplets that make up clouds!

Example : Deinococcus radiodurans

D. radiodurans is by far the best studied specie of this Family. It was originally isolated over 50 years ago from cans of meat treated with large doses of gamma-irradiation during the development of this preservation process. Some cans nevertheless spoiled, and the organism responsible was isolated. Irradiation is now a common method for packaged food preservation, and the dosage used is based on the need to kill this organism, just as autoclaving time and temperature is based on the need to kill endospores. Although involved in food spoilage, D. radiodurans is not pathogenic or itself harmful. D. radiodurans has been a model system for the study of the biochemistry of DNA repair. D. radiodurans cells contain several copies of each of the two chromosomes in a torroidal nucleoid

Thermus & relatives

Diversity
This group is more diverse than are the Deinococci, with 17 species in 4 genera. There are also a large number of partially-characterized isolated. T. aquaticus is by far the best known member of this group, and T. thermophilus, because of its very high growth temperature (up to 85°C as compared with 79°C for T. aquaticus), has also been well-studied.

Metabolism
Thermales are all thermophilic heterotrophs, capable of utilizing a wide range of carbon and energy sources but growing best in media with low concentrations of these organic substrates. These organisms are either obligate aerobes or facultative anaerobes, growing anaerobic by nitrate reduction.

Enzymes
Enzymes from these organisms have proven very useful because of their thermostability. This was demonstrated dramatically in the development of the use of T. aquaticus DNA (Taq) polymerase in automated polymerase chain reaction (PCR). Before this, PCR required the user to manually add DNA polymerase (typically from E. coli) to each sample during each cycle of the reaction, and so PCR remained a tedious and obscure method. The use Taq polymerase, because it is not inactivated during the heat DNA denaturation step in each cycle, allowed the automated cycling of PCR reactions, and now PCR is now the mainstay of molecular biology. The DNA polymease from T. thermophilus is also now widely used, because of its greater thermostability and reverse transcriptase activity. These enzymes have largely been replaced in PCR by DNA polymerases from thermophilic Archaea, which are more processive and accurate (because of their 3´-5´ exonuclease ‘proofreading’ activity), and more thermostable.

These organisms are also the sources of other important thermostable enzymes used in biotechnology and industry. Industrially important enzymes are primarily carbohydrate hydrolases, and are useful because their long lifespan (stability) makes them useful in immobilized enzyme systems.

Enzymes from Thermus have been studied extensively by structural biochemists, because their thermostability (and therefore rigidity at moderate temperatures) often results in the ability to easily grow very uniform crystals for X-ray diffraction analysis and determination of three-dimensional structure. In addition, these enzymes are generally readily over-expressed in E.coli, and easily purified from E. coli extracts; a quick heat-treatment curdles all but the smallest of E. coli proteins, leaving the protein of interest the predominant remainder in solution.

Morphology
Most are filamentous in nature and initially upon isolation, but upon domestication become pleomorphic rods and short filaments. The outer membrane of their Gram-negative-type envelop is loosely attached to the cell wall, appearing corrugated by electron microscopy. In captivity, some cells produce vesicular ‘blebs’ and ‘rotund bodies’, aggregates of cell bound inside a common outer membrane. T. filiformis is a filamentous sheathed specie. Most species produce carotinoid pigments, and so form yellow, orange or red/pink colonies.

Habitat
Thermus and the related genera are readily isolated from neutral pH to slightly alkaline hot springs, at temperatures between 55°-80°C. Halophilic species have been isolated from submarine vents. Hot artificial environments can also harbor Thermus, including thermally polluted water outflows, soil heated by steam pipes, and household hot water heaters.

Example : Thermus aquaticus

The original isolates of T. aquaticus were from Mushroom Spring, Octopus Spring, and other alkaline hot springs in the White Creek area of Yellowstone National Park, in attempt to cultivate the pink filamentous growth that is common in these springs (see Thermocrinus ruber in Chapter 8). It forms pale yellow colonies, growing between 40°C and 79°C, optimally at 70°C. T. aquaticus is an obligate aerobe; it cannot reduce nitrate.

Phylum Chlamydiae (Chlamydia and relatives)



• Phylum Chlamydiae
  • Class Chlamydiae
    • Order Chlamydiales
      • Family Chlamydiaceae
        • Genus Chlamydia
        • Genus Chlamydiophila
        • Genus Clavochlamyia
      • Family Parachlamydiaceae
        • Genus Parachlamydia
        • Genus Neochlamydia
        • Genus Protochlamydia
      • Family Simkaniaceae
        • Genus Simkania
        • Genus Fritschea
      • Family Criblamydiaceae
        • Genus Criblamydia
        • Genus Rhabdochlamydia
        • Genus Piscichlamydia
      • Family Waddliaceae
        • Genus Waddlia

About this phylum

Diversity
Historically, there were only 3 species in this Phylum, all of the genus Chlamydia: the human pathogens C. trachomatis, C. psittaci and C. pneumoniae. (The latter two have ben moved to the genus Chlamydiophila.) Although many new species are now known, this Phylum remains a collection of very closely-related, phenotypically similar organisms. However, environmental surveys using the rRNA PCR approach suggest the diversity both within and without known Families is very much broader than is represented by known species. Although they are sometimes discussed with viruses, because they are obligate intracellular parasites transmitted by small, metabolically inert particles, they are Bacteria phylogenetically and in every other meaningful way.

The Chlamdia are distantly related to Verrucomicrobium, and probably the Planctomycetes as well, all of which have little or no peptidoglycan in their cell walls.

Life cycle
The Chlamydiae are obligate intracellular parasites of eukaryotes with a biphasic life cycle. The ‘elementary body’ (EB) is the infectious phase found in interstitial fluids, secretions, and in the environment. EB’s are small, only 0.2-0.3μm in diameter, and metabolically inert. Although Chlamydiae lack detectable amounts of peptidoglycan, the envelop of EBs is rigid due to heavy disulfide crosslinking of the major outer-membrane protein (MOMP). EBs attach to the host cell surface, probably non-specifically rather than via any specific receptors or adhesins, and endocytosed. The EBs then develop into vegetative ‘reticulate bodies’ (RBs), which metabolize, grow, and divide within the endocytic vesicle. RBs are larger, ca. 1μm cells, and are non-infectious and osmotically fragile, apparently lacking MOMP crosslinking. When the resources of the host cell become limited, most of the RBs differentiate into EBs, which are then released into the surroundings either by host cell lysis or exocytosis.



Metabolism
Although the genomes of Chlamydiae are reduced in animal pathogens to about 1000 genes, they retain the genes required for information processing (transcription, translation, replication), the cell envelop (including peptidoglycan synthesis, even though this is undetectable in practice), and much of central metabolism. Glycolytic enzymes genes are present, as are the genes for an incomplete TCA cycle. The genes for purine and pyrimidine biosynthesis are present in some species, but they seem to rely on the host for most amino acids and cofactors. ATPase, run in ‘reverse’ at the expense of ATP, is probably used to generate the proton gradient required to drive active transport of nutrients from the host cell; no electron transport chain is present.

The Chlamydiae are largely energy parasites. An ATP/ADP antiport is used to acquire ATP from the host an recycle ADP. The ability to synthesize ATP may supplement energy parasitism, or may be required only to generate ADP to supply the ATP/ADP antiport as the cells grow.

Habitat
The Chlamydiae are all obligate intracellular parasites, predominately of animals (as far as we know). The "environmental" Chlamydiae, despite the name, are also obligate intracellular parasites, but they infect protists - especially amoebas. In fact, it may be that amoebas can act as intermediate hosts for the more traditional Chlamydia as well. These environmental species have the same life cycle as the other Chlamydiae, but the elementary bodies float around in the environment instead of the body fluids of the host.

Example species

Chlamydia trachomatis

C. trachomatis is a human pathogen that is the most common venereal disease in the United States - 4 million cases/year. It is easily spread since most infections in females are asymptomatic and untreated. Infection can lead to PID in women, and eventually sterility, and urethritis in men. Repeated ocular infection ("trachoma"), usually in children, leads to blindness, primarily in the third world, and is the leading cause of childhood blindness in the world. Blindness is an indirect result of infection on the inner eyelid; scarring causes the eyelids to curl inwards such that the eyelashes rub painfully across the surface of the eye with every blink. This constant irritation clouds the cornea, obscuring vision. This species also infects the koala, resulting in infertility that, along with habitat loss, is a serious threat to the survival of the species. In fact, the four major infectious diseases of koalas are all chlamydial!

Protochlamydia amoebophila

P. amoebophila grows symbiotically in amoeba of the genus Acanthamoeba. These amoebas are common environmental organisms, although some are opportunistically pathogenic to humans; A. castelliani is commonly found in the tear covering of your eyes, and can cause infections in the eyes, especially those of contact lens wearers. P. amoebophila is a model system for the investigation of the evolution of the human pathogen Chlamydiae. The genome of P. amoebophila less reduced than that of the pathogenic Chlamydiae. At about 2.4MBP in length, with over 2000 protein-encoding genes, it is twice the size as those of the pathogenic Chlamydiae, and as large as those of many free-living Bacteria. It has a complete TCA cycle, from which is can synthesize glycine, serine, glutamine, and proline. Unlike pathogenic Chlamydiae, it cannot synthesize tryptophan; this ability is a virulence factor in the pathogens. P. amoebophila has an abbreviated electron transport chain, which is probably used to generate a proton gradient for active transport, but may also be used to generate ATP by oxidation phosphorylation to supplement that acquired from the host.

Phylum Planctomycetes (Planctomyces & relatives)



• Phylum Planctomycetes
  • Class Planctomycetacia
    • Family Planctomycetales
      • Genus Planctomyces
      • Genus Gemmata
      • Genus Isosphaera
      • Genus Pirellula
      • Genus Blastopirellula
      • Genus Rhodopirellula
      • Genus Pirella
      • Genus Singulisphaera
      • Genus Nostocoida
        
        
• Incertae sedis
  • Genus Brocadia
  • Genus Kuenenia

About this phylum

Diversity
The diversity of the Planctomycetes is unclear; although they are morphologically conspicuous, because they divide by budding, are stalked and often form spectacular rosettes, they are rarely isolated in pure culture for study. The Phylum is distantly but specifically related to the Chlamydiae and Verrucomicrobia.

Metabolism
Cultivated Planctomycetes are all aerobic mesophilic oligotrophic heterotrophs, except Isosphaera pallida, which is moderately thermophilic (up to 55°C), and Brocadia anammoxidans and Kuenenia stuttgartiensis, which carry out the anaerobic ammonia oxidation. The heterotrophs are capable of growth on a wide range of sugars and sugar derivatives, including polysaccharides. All lack peptidoglycan, and are resistant to β-lactam antibiotics such as ampicillin; this trait can be useful in enrichment cultures and isolations.

Morphology
Typically these organisms are cocci or oval-shaped. Most have stalks, but these can be too thin or short to be apparent by light microscopy. These stacks are external fibrous structures, unlike the cytoplasmic extensions of appendaged Bacteria. Stalked forms often form planktonic rosettes. Fimbrae are common, and newly-released buds are often flagellated and highly motile. They lack the peptidoglycen cell wall, but have an external pitted wall of unknown composition. The nucleoids of Planctomycetes are quite distinct and condensed.



All of the members of the Planctomycetes contain internal membrane-defined compartmentalization. The cytoplasm is divided into the riboplasm, containing ribosomes and DNA, and the paryphoplasm, that contains RNA (of unknown type or function) but not ribosomes. These compartments are separated by a membrane; the internal compartment contains the riboplasm and nucleoid and is termed the pirellulosome. In Gemmata, the nucleoid is separated from most of the riboplasm by an additional double-membrane ‘nuclear’ envelope. This "nucleus" is very different than those of eukaryotes, however, in that it contains apparently functional ribosomes. In the Brocadia (and presumably Kuenena), there is an additional membrane separating the ‘anammoxisomes’ from the rest of the cell. This membrane has an unusual lipid composition specially designed to keep the toxic intermediates of this reaction contained.

Habitat
Most Planctomycetes are aquatic, and appear most commonly in eutrophic environments. However, Isosphaera is found in the phototrophic mats of hot springs (35°C-55°C), Brocadia and Kuenenia are found in anaerobic waste digesters, and ssu-rRNA sequences from this group are isolated from a wide range of environments.

Example species

Blastopirellula marina

Blastopirellula (previously Pirellula) marina is a relatively common freshwater specie, oval with short stalks and forms small, flower-like rosettes (as compared with the spectacular ‘fireworks’ rosettes of Planctomyces species). Blastopirellula has the simplest form of compartmentalization of the Planctomycetes. The intracytoplasmic membrane (ICM) is a simple ovoid separating the riboplasm, containing both the ribosomes and the nucleoid, from the paryphoplasma. The paryphoplasma is primarily at one end of the cell, i.e. it is polar.

Isosphaera pallida

Isosphaera pallida is an unusual member of this group phenotypically; individual cells are cocci, but these form filamentous chains that contain gas vacuoles and are motile by gliding. Budding occurs along the axis of the chain, and so daughter cells are formed interstitially. Isolated from hot spring phototrophic mats, it was originally mistaken for a cyanobacterium. I. pallida has a cell structure very similar to that of Planctomyces, with a single membrane (ICM) separating the cytoplasm into paryphoplasm and riboplasm. The paryphoplasm is highly polar, excluded mostly to one end of the cell as a sort of vesicle, but it still forms a thin layer between the cytoplasmic membrane and the ICM all the way around the cell.

Brocadia anammoxidans

Brocadia anammoxidans is phenotypically very different than the other Planctomycetes; it is an anaerobic autotroph, gaining energy by the production of dinitrogen gas by the reaction of ammonia and nitrite; this is known as anaerobic ammonia oxidation, or the ‘anammox’ reaction. Carbon fixation is by the acetyl-CoA pathway. B. anammoxidans has a bit more internal complexity than most Planctomycetes, but is based on the same cell structure as the previous examples. The paryphoplasm is a relatively thin layer all around the cell (not polarized), and there is an additional membrane-bound structure, the anammoxisome. This is a critical aspect of the anammox reaction, which includes a very highly reactive intermediate, hydrazine (a.k.a. rocket fuel). Note that the anammoxisome membrane is not attached to or part of the ICM, nor is the ICM attached to or part of the cytoplasmic membrane (this is true of all of the planctomycetes).

Gemmata obscuriglobus

Gemmata obscuriglobus spherical or ovoid non-stalked specie. Previously thought to have a large indentation in the surface of cell, seen in many electron microscopic images, this seems to be an artifact of dehydration in preparation for microscopy. Gemmata is the most complex Planctomycete in internal structure. As in Brocadia (above), the paryphoplasm forms a relatively thin layer all around the cell, between the CM and the ICM. In Gemmata, however, there is an additional double-layered membrane within the riboplasm surrounding the nucleoid. This ‘nuclear envelop’ is studded on both sides with ribosomes, and continuous with the cell membrane. Openings in this membrane allows movement of riboplasm contents between two compartments.

What is the difference between paryphoplasm and periplasm?

A substantial issue with these cellular structures in Planctomycetes is distinguishing the paryphoplasm from typical Gram-negative periplasm. These organisms have a cell wall outside the cell membrane, but it is not a peptioglycan (what it is, is not known). They do not have a membrane outside of the cell wall; they lack the hallmark Gram-negative outer membrane. Or do they? What what’s being called the ICM (internal cellular membrane) is really the cytoplasmic membrane, and what’s being called the CM (cytoplasmic membrane) is really the outer membrane? In this case, the riboplasm becomes traditional cytoplasm, and the paryphoplasm becomes periplasm (sometimes pretty substantial, as in Thermotoga). The difference, then, with Planctomycetes would be that they lost the peptidoglacan cell wall, and reinvented it outside of the outer membrane, much like the S-layer of many Bacteria and Archaea.

However, even if this is the case (and the authorities on Planctomycetes argue it is not) that doesn't change the most interesting observation, that Gemmata has a sort of nucleus with a nuclear envelop. Yes, it has ribosomes in it, but maybe eukaryotic nuclei do, too (involved in nonsense-mediated decay), and surely there is some sort of functional differentiation between riboplasm inside and outside the nuclear envelop. Just for example, the ribosomes translating outside of the "nucleus" cannot be translating mRNAs that are still being transcribed; this lack of linkage between transcription and translation is usually cited as an important distinction between ‘prokaryotes’ (this misguided term is discussed in detail in Chapter 2) and eukaryotes.

Reductive evolution in parasites

It is common for parasites, especially endoparasites, to evolve by simplification, and the more the parasite relies on it's host for the things it needs, the more it can simplify. This ‘reductive evolution’ (which used to be called ‘degeneration’, or sometimes ‘devolution’, thus the name of the inexplicably popular band of the early 80's, ‘Devo’) allows the parasite to focus it's resources on reproduction. For example, many parasitic worms lack digestive systems (they absorb their nutrients directly through their cuticle), circulatory system, &c, &c, and in extreme cases are little more than genitals than hook onto the GI tract (or elsewhere) of their host. Even ectoparasites often become simplified; there are a slew of ectoparasites that are little more than stomachs, sucking mouths, and reproductive organs (i.e. leeches).

Even bacterial parasites can evolve by simplification, good examples being the Chlamydiae, Rickettsiae, and Mycoplasmas. Perhaps more extreme examples would be plastids and mitochondria, and some viruses may have originated by simplification of cellular intracellular parasites. In Bacteria, this simplification is most easily seen in their genomes; the sizes of the genomes of these obligate intracellular parasites has been drastically reduced. Any gene that can be done without is eliminated. The genomes of the human pathogenic Chlamydia are only 1Mbp; about a thousand genes, only 1/4th as big as wild E. coli and about 1/2 that of the smallest genomes of free-living Bacteria. The reasons for simplification are many; in addition to the usual reasons for simplification in parasites generally, the smaller the genome, the faster you can replicate it, and the simpler the organism, the faster it can evolve. The latter is especially important for a bacterial parasite, which is in a continuous arms race with it's host.

Questions for thought

• If you were interested in getting some novel isolates of Deinococcus or Thermus, where would you look and how would you set up the enrichment cultures?
  
  
• Can you think of any enzymes other than DNA polymerases from thermophilic organisms that might be useful? What about enzymes from organisms that live in extremely cold environments? Saline environments?
  
  
• How far do you think a bacterial intracellular parasite could minimize itself? Why not further? What could or could not it do without?
  
  
• What is the difference between a minimized, obligately intracellular bacterial parasite like Chlamydiae and a virus?
  
  
• The Chlamydiae we know about infect animals (mammals, birds, reptiles and insects) and amoebas. Where would you look for whole new kinds of Chlamydiae? How would to go about this?
  
  
• Gemmata has an double-membrane around its nucleoid. How would you determine how much this membrane is like the nuclear envelop of eukaryotes?
  
  
• How would you go about determining whether or not the paryphoplasm was similar to the periplasm of other organisms?
  
  
• Many bacteria divide, like Planctomycetes, by budding rather than by binary fission. What are the ramifications of this for both the mother and daughter cells, compared to binary fission?
  
  
• Planctomycetes divide by budding, and Gemmata has a double-membrane enclosed nucleoid. Draw on a peice of paper how you imagine cell division working in this organism, given that even early buds have a ‘nucleus’. How is your scheme similar to and different from the eukaryotic cell cycle?

Previous or Next lecture